Search

Search Results

1 results found. Results per page: [ 20 ][ 40 ][ 60 ][ 80 ][ 100 ][ 200 ][ 300 ]

Sort by: [ Publication Date ][ Score ]

Year of publication: [ 2025 ][ 2024 ][ 2023 ][ 2022 ][ 2021 ][ 2020 ][ 2019 ][ 2018 ][ 2017 ][ 2016 ][ 2015 ][ 2014 ][ 2013 ][ 2012 ][ 2011 ][ 2010 ][ 2009 ][ Any ]

Direction: [ Desc ][ Asc ]

• Research Paper Volume 11, Issue 2 pp 523-535

  lncRNA-ES3/miR-34c-5p/BMF axis is involved in regulating high-glucose-induced calcification/senescence of VSMCs

  Relevance score: 11.611233

  Xiao Lin, Jun-Kun Zhan, Jia-Yu Zhong, Yan-Jiao Wang, Yi Wang, Shuang Li, Jie-Yu He, Pan Tan, Yi-Yin Chen, Xue-Bin Liu, Xing-Jun Cui, You-Shuo Liu

  Keywords: miR-34c-5p, lncRNA-ES3, BMF, VSMCs calcification/senescence, vascular aging, diabetes

  Published in Aging on January 17, 2019

  Show abstract
  Hide abstract

  Vascular calcification/aging is common in diabetes and is associated with increased morbidity and mortality of patients. MiR-34c-5p, not miR-34c-3p, was suppressed significantly in calcification/senescence of human aorta vascular smooth muscle cells (HA-VSMCs) induced by high glucose, which was proven by the formation of mineralized nodules and staining of senescence associated-β-galactosidase staining (SA β-gal) positive cells. Overexpression of miR-34c-5p alleviated calcification/senescence of HA-VSMCs, whereas inhibition of miR-34c-5p received the opposite results. Bcl-2 modifying factor (BMF) was a functional target of miR-34c-5p and it was involved in the process of calcification/senescence of HA-VSMCs. Besides, lncRNA-ES3 acted as a competing endogenous RNAs (ceRNA) of miR-34c-5p to enhance BMF expression. Further, lncRNA-ES3 inhibited miR-34c-5p expression by direct interaction and its knockdown suppressed the calcification/senescence of HA-VSMCs. Our results showed for the first time that the calcification/senescence of VSMCs was regulated by lncRNA-ES3 /miR-34c-5p/BMF axis.

  

  (A) HA-VSMCs were treated with NG, OC, or HG for 14 days and then subjected to Alizarin Red S staining. The calcium content was extracted with cetylpyridinium chloride and quantified by spectrophotometry. Representative pictures are shown and the scale bar is 100 μm. (B) HA-VSMCs were treated with NG, OC, or HG for 72 hours and then subjected to SA-β-gal staining. Semi-quantitative analysis of SA-β-gal positive cells were performed using Image J. (C and D) qRT-PCR showing the expression of miR-3c-5p and miR-34c-3p in the above three groups. The data are expressed as mean ± SD, n=3, **p<0.005, ***p<0.0005. NG: normal glucose; OC: osmolarity control; HG: high glucose.

  
  
  

  

  (A) HA-VSMCs was transfected with miRNA NC, miR-34c-5p mimics, and miR-34c-5p inhibitor, and subjected to qRT–PCR analysis of miR-34c-5p. (B–D) HA-VSMCs were transfected with miRNA NC, miR-34c-5p mimics, and miR-34c-5p inhibitor, respectively. Then, ALP activity, OC secretion, and Runx2, p16, and p21 protein levels were measured. The data are expressed as mean ± SD, n=3, *p<0.05, **p<0.005, ***p<0.0005. NC: negative control.

  
  
  

  

  (A) Schematic representation of the putative binding sites between lncRNA-ES3 and miR-34c-5p, and the mutant sites in Mut-lncRNA-ES3 reporter were underlined. (B) qRT-PCR showed the expression of lncRNA-ES3 in HA-VSMCs cultured with NG, OC, and HG. (C) HA-VSMCs was transfected with miRNA NC and miR-34c-5p mimics and harvested for the examination of lncRNA-ES3 by qRT-PCR. (D) The inhibitory efficiency of shRNAs targeting lncRNA-ES3 was verified by qRT-PCR. (E) HA-VSMCs were transfected with shRNA NC and shRNA-ES3-2, and the expression of miR-34c-5p was detected by qRT-PCR. (F) The WT-lncRNA-ES3 3’UTR and the Mut-lncRNA-ES3 3’UTR reporters were co-transfected with miR-34c-5p mimics or control oligos into HA-VSMCs. Forty-eight hours after transfection, luciferase activities were measured. (G) The expression of lncRNA-ES3 was detected by qRT-PCR after biotin-labeled miR-34c-5p pull-down assay. (H) RIP and qRT-PCR assays were performed to explore the binding efficiency of miR-34c-5p and lncRNA-ES3 to Ago2 protein in HA-VSMCs. The data are expressed as mean ± SD, n=3, *p<0.05, **p<0.005, ***p<0.0005. NG: normal glucose; OC: osmolarity control; HG: high glucose; NC: negative control.

  
  
  

  

  (A) Schematic representation of the miR-34c-5p putative target sites in BMF 3’UTR and alignment of miR-34c-5p with WT and Mut BMF 3’UTR showing pairing. The mutated nucleotides were underlined. (B and C) HA-VSMCs were transfected with miRNA NC, miR-34c-5p mimics, and miR-34c-5p inhibitors, and harvested for the examination of BMF mRNA and protein. (D) The WT-BMF 3’UTR and Mut-BMF 3’UTR were co-transfected with miR-34c-5p mimics or control oligos into HA-VSMCs. Forty-eight hours after transfection, luciferase activities were measured. (E and F) qRT-PCR and Western blot analysis showed the expression level of BMF in HA-VSMCs cultured with NG, OC, and HG. (G) HA-VSMCs were transfected with shRNAs NC and shlncRNA-ES3-2, and the protein level of BMF was detected by Western blot. The data are expressed as mean ± SD, n=3, *p<0.05, **p<0.005, ***p<0.0005. NG: normal glucose; OC: osmolarity control; HG: high glucose; NC: negative control.

  
  
  

  

  (A and B) The ALP activity and OC secretion were detected in HA-VSMCs with different treatment, respectively. (C) Representative images of Western blot analyses of p16, p21, Runx2, and BMF in HA-VSMCs with different treatment are shown. (D and E) Alizarin Red S staining showed the mineralized nodules in HA-VSMCs, and the calcium content was extracted with cetylpyridinium chloride and quantified by spectrophotometry. Representative pictures are shown and the scale bar is 100 μm. (F and G) SA-β-gal staining showed the senescent cells of HA-VSMCs with different treatment, and the quantification of SA-β-gal-stained positive cells is shown. Representative pictures are shown and the scale bar is 100 μm. (H) The model proposed to explain the mechanism of miR-34c-5p in inhibiting VSMC calcification/senescence is shown. Herein, lncRNA-ES3 inhibits miR-34c-5p expression, enhances the expression of BMF, and finally promotes calcification/senescence of VSMCs. The data are expressed as mean ± SD, n=3, *p<0.05. NG: normal glucose; HG: high glucose; NC: negative control.